High performance liquid chromatography (HPLC) is a technique that has been used for many years as a means of separating, identifying, purifying and quantifying components of often complex mixtures. HPLC is an important tool used by biotechnological, biomedical, and biochemical research as well as in the pharmaceutical, cosmetics, energy, food, and environmental industries.
Conventional HPLC typically is performed using chromatographic columns with inside diameters (I.D.'s) in the range of about 2-10 mm, 4.6 mm columns being a common standard. Microcolumn liquid chromatography (“LC”), which is the most widely accepted term to describe liquid chromatography using packed columns having I.D.'s of 2mm or less, is gaining in popularity. Advantages of microcolumn LC include the ability so analyze smaller sample volumes, reduction of solvent usage, and enhanced mass sensitivity.
Due to their relatively large volume, sample injection systems developed for conventional HPLC systems are inadequate for use in microcolnum LC systems. Sample volume requirements for microcolumn LC relative to conventional HPLC can be determined by considering a constant sample volume to column volume ratio. For example, a direct scaling of injection volumes indicates that a 10 μL sample injection into a 4.6 mm i.d. conventional HPLC column would be equivalent to 43 nL, 4.7 nL and 1.2 nL sample injections into 300 μm, 100 μm and 50 μm i.d. nanobore HPLC columns, respectively.
Injection valves and injection methods have been developed in an attempt to meet the demands of volume injections of 500 nL and less. See, for example, those disclosed in Vissers, J. P. C., Arnoud, H. R., Ursem, M. and Chervet, J. P., “Optimised injection techniques for micro and capillary liquid chromatography”, J. of Chrom A, 746, p 1, (1996); Bakalyar, S. R., Phipps, C., Spruce, B. and Olsen, K., “Choosing sample volume to achieve maximum detection sensitivity and resolution with high-performance liquid chromatography columns of 1.0, 2.1 and 4.6 mm I.D.”, J. Chrom. A, 762, p 167, (1997); Foster, M.D., Arnold, M. A., Nichols, J. A. and Bakalyar, S. R., “Performance of experimental sample injectors for high-performance, liquid chromatography microcolumns”, J. Chrom. A, 869, p 231, (2000). Others include commercially available valves from companies such as VICI Valco Instruments, Rheodyne and Upchurch Scientific. Valve designs include both external and internal sample loops. Injection volumes of less than 100 nL are typically achieved using valves with internal sample loops where a groove in the rotor serves as the loop. Larger injection volumes can be achieved with either internal loops or external loops connected to the valve ports.
In conventional HPLC systems, the resolution and efficiency of the separation have been primarily determined by the performance of the column itself. In contrast to conventional HPLC systems, the resolution and separation efficiency of microscale HPLC systems is often determined by band-broadening from the sample injector, connection tubing and detector cell.
The band-broadening due to the instrumental components as well as the column is called dispersion and is characterized by the variance of the peak shape. The ideal injection (square pulse) will introduce a variance, σ2, of ˜V2/12, where V is the injection volume. In practice, this ideal injection performance has not been demonstrated for injection volumes of <500 nL and the sample is contained in a volume much larger than the ideal square pulse.
In addition to minimizing the variance, microcolumn LC systems must have the ability to quantitatively inject small sample volumes. Although internal loop injectors can directly provide for injection volumes as small as 10 nL, the absolute accuracy and consistency from loop to loop are poor.
As the injection volumes become smaller, quantitative injections become more difficult. This difficulty is reflected in the literature as well as product specifications of commercial instruments. For example, the specifications for the relative standard deviation on the Agilent 1100 HPLC system using a conventional injection size of 5 μL is 6 times better than for the Agilent 1100 CapLC using injection volumes of 0.2-1.0 μL. No specification is given for smaller injection volumes. Typical repeatability for peak areas in microscale LC are in the 2-6% range compared to 0.5% for conventional size systems.
Accordingly, there is a need in the art for an HPLC injector and a method for injecting a sample fluid into a separation column that provides the ability to reduce injection variance and maintain very accurate and precise injection volumes.